A γ-Lactam Siderophore Antibiotic Effective against Multidrug-Resistant Gram-Negative Bacilli

Abstract

Treatment of multidrug-resistant Gram-negative bacterial pathogens represents a critical clinical need. Here, we report a novel γ-lactam pyrazolidinone that targets penicillin-binding proteins (PBPs) and incorporates a siderophore moiety to facilitate uptake into the periplasm. The MIC values of γ-lactam YU253434, 1, are reported along with the finding that 1 is resistant to hydrolysis by all four classes of β-lactamases. The druglike characteristics and mouse PK data are described along with the X-ray crystal structure of 1 binding to its target PBP3.

Figure 1.

Comparator agents and select siderophore containing PBP inhibitors as well as the β-lactamase inhibitor avibactam. Highlighted in red is the site that covalently modifies the PBP or β-lactamase catalytic serine. When a siderophore mimic is present, it is highlighted in blue.

Figure 2.

Minimum inhibitory concentrations (MICs) of 1 against P. aeruginosa (197 clinical isolates), K. pneumoniae (100 clinical isolates), and E. coli (100 clinical isolates).

Figure 3.

Mean plasma concentration–time profile of 1 after IV (50 mg/kg) administration in mice.

Figure 4.

Determination of the IC₅₀ for P. aeruginosa PBP3 using a competitive assay. Bocillin, a fluorescent substrate of PBP3, was reacted with an enzyme that had been pre-incubated with increasing concentrations of 1. The IC₅₀ was calculated as the concentration of 1 required to reduce the fluorescence intensity of the bocillin-labeled protein by 50%.

Figure 5.

DSF assay of 1 and ceftazidime binding to P. aeruginosa PBP3. The derivative of the change in fluorescence is plotted versus temperature.

Figure 6.

Electron density of 1 in the active site of P. aeruginosa PBP3. (A) Unbiased electron difference density (|F_o|–|F_c|) showing the density for a covalently bound 1 in the active site. Density is contoured at the 3σ level. Prior to map calculation, 10 cycles of Refmac refinement were carried out with 1 removed from the structure. The S349 side chain was observed in two conformations (labeled 1 and 2, refined with occupancies of 0.6 and 0.4, respectively). 1 is shown with cyan-colored carbon atoms. (B) Same as in (A) but the view is rotated ~100° around the x axis. (C) Stereodiagram of the 2|F_o|–|F_c| density map of the active site region; density is contoured at the 1σ level. PDB ID 6VOT.

Figure 7.

Interactions of 1 in the active site of P. aeruginosa. PBP3. Hydrogen bonds are depicted as black dashed lines. Water molecules are shown as round red spheres. Key active site residues are labeled.

Figure 8.

Comparison of the 1 and ceftazidime (4)-bound structures of P. aeruginosa PBP3. (A) Superposition of the PBP3–1 complex (gray protein carbon atoms) onto the PBP3–ceftazidime structure (gold-colored carbon atoms). The ligands are depicted in a ball-and-stick model, and 1 is shown with cyan carbon atoms. (B) Same as in (A) but a close-up view of the active site region near the covalent bond with S294 to highlight differences in the β-lactam (ceftazidime, 4)- and γ-lactam (1)-based ligands. Hydrogen bonds of 1 and ceftazidime with their corresponding PBP3 coordinates are shown as black and gray dashed lines, respectively.

Scheme 1.. Synthesis of YU253434, 1^a

^a(a) Allyl 2-(diethoxyphosphoryl)acrylate, DCM, RT, 1 h, 98%; (b) tert-butyl 2-chloro-2-oxoacetate, DCM, 0 °C to RT, Hunig’s base, 18 h, 100% crude; (c) acetonitrile, triethylsilane, Pd(PPh₃)₄, 0 °C to RT, 65%; (d) (i) oxalyl chloride, 12, (ii) MSTFA, Hunig’s base, 13, 38%; (e) 2:1 DCM/TFA, triethylsilane, 0 °C to RT, 3.5 h, 100% crude; (f) (i) oxalyl chloride, catalytic DMF, 16, (ii) MSTFA, Hunig’s base, 15, 3.5 h, 100% crude; (g) 2:1 DCM/TFA, triethylsilane, 0 °C to RT, 1.5 h, toluene chase, reverse-phase MPLC C18, 0 to 60% acetonitrile 0.1% formic acid/water with 0.1% formic acid, 38%.